Thermal dependency of RAG1 self-association properties.

BACKGROUND: Functional immunoglobulin and T cell receptor genes are produced in developing lymphocytes by V(D)J recombination. The initial site-specific DNA cleavage steps in this process are catalyzed by the V(D)J recombinase, consisting of RAG1 and RAG2, which is directed to appropriate DNA cleavage sites by recognition of the conserved recombination signal sequence (RSS). RAG1 contains both the active site and the RSS binding domains, although RAG2 is also required for DNA cleavage activity. An understanding of the physicochemical properties of the RAG proteins, their association, and their interaction with the RSS is not yet well developed. RESULTS: Here, we further our investigations into the self-association properties of RAG1 by demonstrating that despite the presence of multiple RAG1 oligomers, only the dimeric form maintains the ability to interact with RAG2 and the RSS. However, facile aggregation of the dimeric form at physiological temperature may render this protein inactive in the absence of RAG2. Upon addition of RAG2 at 37 degrees C, the preferentially stabilized V(D)J recombinase:RSS complex contains a single dimer of RAG1. CONCLUSION: Together these results confirm that the functional form of RAG1 in V(D)J recombination is in the dimeric state, and that its stability under physiological conditions likely requires complex formation with RAG2. Additionally, in future structural and functional studies of RAG1, it will be important to take into account the temperature-dependent self-association properties of RAG1 described in this study.

DNA cleavage of a cryptic recombination signal sequence by RAG1 and RAG2. Implications for partial V(H) gene replacement.

Antibody and T cell receptor genes are assembled from gene segments by V(D)J recombination to produce an almost infinitely diverse repertoire of antigen specificities. Recombination is initiated by cleavage of conserved recombination signal sequences (RSS) by RAG1 and RAG2 during lymphocyte development. Recent evidence demonstrates that recombination can occur at noncanonical RSS sites within Ig genes or at other loci, outside the context of normal lymphocyte receptor gene rearrangement. We have characterized the ability of the RAG proteins to bind and cleave a cryptic RSS (cRSS) located within an Ig V(H) gene segment. The RAG proteins bound with sequence specificity to either the consensus RSS or the cRSS. The RAG proteins nick the cRSS on both the top and bottom strands, thereby bypassing the formation of the DNA hairpin intermediate observed in RAG cleavage of canonical RSS substrates. We propose that the RAG proteins may utilize an alternative mechanism for double-stranded DNA cleavage, depending on the substrate sequence. These results have implications for further diversification of the antigen receptor repertoire as well as the role of the RAG proteins in genomic instability.

Self-association and conformational properties of RAG1: implications for formation of the V(D)J recombinase.

RAG1 and RAG2 catalyze the initial DNA cleavage steps in V(D)J recombination. Fundamental properties of these proteins remain largely unknown. Here, self-association and conformational properties of murine core RAG1 (residues 384-1008) were examined. As determined by multi-angle laser light scattering measurements, the molecular masses of two predominant core RAG1 species corresponded to dimeric and tetrameric states. Similar results were obtained using a RAG1 fragment containing residues 265-1008, indicating that a non-core portion of RAG1 does not alter the oligomerization states observed for the core region. The fraction of core RAG1 in the tetrameric state increased significantly at lower ionic strengths (0.2 versus 0.5 M NaCl), indicating that this oligomeric form may factor into the physiological function of RAG1. In addition, the secondary structural content of core RAG1, obtained by circular dichroism spectroscopy, demonstrated a significant dependence on ionic strength with a 26% increase in alpha-helical content from 0.2 to 1.0 M NaCl. Together, these results indicate that structural and oligomerization properties of core RAG1 are strongly dependent on electrostatic interactions. Furthermore, the secondary structure of core RAG1 changes upon binding to DNA, with larger increases in alpha-helical content upon binding to the recombination signal sequence (RSS) as compared with non-sequence-specific DNA. As shown by electrophoretic mobility shift assays, higher order oligomeric forms of core RAG1 bound to the canonical RSS. Furthermore, core RAG2 (residues 1-387) formed complexes with multimeric RAG1 species bound to a single RSS, providing additional support for the physiological relevance of higher order oligomeric states of RAG1.